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Vejendla.Ravikumar* et al. /International Journal Of Pharmacy&Technology
IJPT | March-2011 | Vol. 3 | Issue No.1 | 1433-1448 Page 1433
ISSN: 0975-766X Available Online through Research Article
www.ijptonline.com DEVELOPMENT OF RP-HPLC METHOD FOR THE QUANTIFICATION OF KARANJIN I N THE
SEED EXTRACTS OF PONGAMIA GLABRA Vejendla.Ravikumar*, Md.SirajuddinKhan 1, M.Manoj kumar 2
1Departmant of Pharmacy, Sri Indu Institute of Pharmacy, Hyderabad. 2Departmant of Pharmacy, Sir C.R.Reddy College of Pharmaceutical Sciences, Eluru.
Email: [email protected]
Received on 19-12-2010 Accepted on 01-01-2011
ABSTRACT:
The main aim of the work, done by the author, is to estimate the active ingredient Karanjin, a cyclic furano-
flavonoid, present in the different parts of seed extracts of karanja (Pongamia glabra) mentioned the structure in
Fig.1. and to carry out the Quantitative Analysis of the Karanjin. Reporting the % of Karanjin present in the
different solvent extracts namely: ethyl acetate, methanol & hexane of dry seed, kernel, shell and fresh seed &
kernal of Pongamia glabra. By taking 200g. of each part found that in dry kernel maximum amount of 36.92g. is
extracted by using methanol solvent, which was quantified by using HPLC was reported as 18.46% which is
maximum when compared with other parts or by using other solvents.
OO
O
OCH3
Fig1. Structure of Karanjin
INTRODUCTION 1:
The author thought to discuss some of the important features of flavonoid compounds as the present
phytochmical work carried out was mainly concern with the flavonoid compounds.
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Flavonoids are yellow pigments, which occur in plant kingdom either in the free state or as glycosides
or associated with tannins. These are also known as the anthoxanthins. Chemically, the flavones are hydroxylated
derivative of flavone (2-phenyl-4-chromone), which are partially alkylated. In most of the flavones, positions 5
and 7 are hydroxylated and also one or more of positions 3, 4, 5 are also hydroxylated. Further, positions 31 and
51 are often methylated whereas positions 5, 7 and 41 are usually unmethylated.
Figure: 2. Basic structure of flavone, flavonol, and flavanone (flavonoids). Quercetin is a flavonol with OH
(hydroxy) group at 3, 5, 7, 31, 41 positions. Kaempferol is a flavonol with OH group at 3, 5, 7, 41 positions.
Quercetagetin is a flavonol with OH group at 3, 5, 6, 7, 31, 41 position.
Flavones show two absorption bands, one at 330-350 nm and other at 250-270 nm. Thus, it becomes
possible to distinguish flavones from the anthocyanins on the basis of absorption bands and also by colour
reactions.
PROPERTIES OF FLAVONOIDS: 1
• Many flavones are yellow solids
• Mostly flavones are soluble in water, ethanol, dilute acids and alkalis.
• Flavones are precipitated by lead salt.
• With ferric chloride, flavones give either a dull green or a red brown colour.
• Shinoda test: Alcoholic solution of flavones in the presence of magnesium ribbon and concentrated
hydrochloride gives magenta colour.
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PHARMACOLOGICAL ACTIVITIES OF FLAVONOIDS:
Flavonoids are phenolic compounds with nuclei arranged in a C6 - C3 - C6 configuration. They have
several beneficial properties for plants. The co-evolution of plants and insects has resulted in different plant
defense mechanisms, one of which is the accumulation of phytochemicals, which may affect feeding, or growth
of insects.1 For example, the resistance of soya bean, Glycine max L., to the cabbage looper, Trichoplusia ni,
appears to be due to the presence of the leaf flavonoids, daidzein, glyceolin, sojagol and coumestrol.2 The
generalist feeder, Spodoptera frugiperda, avoids plants known to contain flavone.3 Similarly, the C-glycosyl
flavone (maysin) in maize silk has antibiotic activities against corn earworm larvae, Helicoverpa zea (Boddie).4
Flavonoids also function to shield plants from UV radiation, act as signaling molecules in plant bacterium
symbioses, and are the primary pigments that attract pollinators and seed dispersers5 although flavonoids are
widely distributed among the flowering plants, particular classes of flavonoids have distinct functions in different
plant groups. For example, flavonols are essential for male fertility in maize and petunia6, but they do not appear
to have a similar function in Arabidopsis.7
Isoflavonoids are the major phytoalexins in legumes8, whereas 3- deoxyanthocyanidins fulfill similar
functions in Sorghum bicolor and other grasses9 multiple mechanisms have been proposed to explain the diversity
of phytochemicals between different plants.10,11
From a pharmaceutical perspective, flavonoids possess a remarkable spectrum of biochemical and
pharmacological activities. Different flavonoids exhibit antioxidative, antibacterial, anti-inflammatory,
antiallergic, antimutagenic, antiviral, antineoplastic, anti-thrombotic, and vasodilatory properties.12,13Catechin and
catechin derivatives, oligomeric proanthocyanidins, quercetin and quercetin chalcone, Ginkgo flavone glycosides,
silymarin, and others are utilized in the prevention and treatment protocols for cardiovascular disease, cancer,
asthma, periodontal disease, liver disease, cataracts and macular degeneration.14-18 Kuntz et al19 reported that
dietary polyphenols could have a significant role in the prevention of colon cancer by blocking hyper-
proliferation of the epithelium and by promoting apoptosis.
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BOTANICAL CHARACTERISTICS AND SIGNIFICANCE OF P. GLABRA:
TAXONOMICAL CLASSIFICATION: [20]
Kingdom : plantae (plants)
Sub kingdom : tracheobionta (vascular plants)
Super division : spermatophyte (seed plants)
Division : magnoliophyta (flowering plants)
Class : liliopsida (monocotyledons)
Sub class : arecidae
Order : arecales
Family : leguminacea
Genus : Pongamia
Species : glabra / pinnata
VERNACULAR NAMES: [21]
Telugu : Gaanuga, Pungu
Tamil : Ponga, Pongam.
Oriya : Koranja.
Kanada : Honge.
Assam : Karchaw.
USES OF PLANT PONGAMIA: [22]
Traditional Uses:
� The wood of the plant is used in building material, ploughs, combs, yokes, oil-mills and solid wheel of a
cart and as fuel.
� The oil extract from the seeds is used in lamps by the weaker section of the society and also in soap
making.
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� Seeds are used as fish poison.
� Leaves are used as fodder, as manure for rice and sugarcane fields.
Pharmacological Uses:
� The fresh bark of P.pinnata is used internally to cure bleeding piles.
� The root and the bark are bitter, anthelmintic and used in vaginal and skin diseases.
� A poultice of the leaves is applied on ulcers infected with worms.
� Seeds are anthelmintic, bitter, acrid and carminative they are useful in inflammations, pectoral diseases,
chronic fever, hemorrhoids and anemia.
� The oil is styptic, anthelmintic, good in leprosy, piles, ulcers, chronic fever and pain in liver.
� A decoction of dried flowers is given for diabetes.
� The juice of the roots is used for cleaning teeth, strengthening gums, against gonorrhea and cleaning foul
ulcers.
� The juice of the plant is used for treating diarrhea, cough, leprosy and gonorrhea.
� Aqueous extract of stem bark exhibits significant CNS sedative and antipyretic activities.
MATERIALS:
All chemicals were analytical grade: Methanol from Qualigens fine chemicals (Mumbai, India), Millipore-
Water used for the preparation of mobile phase solutions, was obtained from All Quartz Double Distilled, Bhanu
Scientific Instruments Company Pvt. Ltd. (Bangalore, India). All the above solutions were de-gassed in an
ultrasonic bath (Sonicator), for 30min.
Stock solution of karanjin for generating standard curves were prepared by dissolving 1.2mg of compound
in 20ml s.v.f with methanol to yield concentration of 6000mg/ml. Working standard solution of karanjin obtained
by diluting 1, 0.8, 0.6, 0.4, 0.2, 0.1ml in 10ml standard volumetric flask so as to get 60, 48, 37, 24, 12, 6µg/ml
concentration of karanjin respectively to get a calibration curve
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Chromatography was performed on HPLC equipment consisting of LC-10AS pumps, SPD – 10A UV-
Visible detector, and an injector equipped with a 20ul sample loop (Rheodyne, USA). Analytical separation was
on column packed with 3µm Waters LC - 18 stationary phase (Waters, USA). The dimension of the separation
column was 150 x 4.6mm i.d. with 3um particle size. Data and chromatograms were collected using C-7RA
chromatopac software system (Shimadzu, Japan). Dissolution of the compound was enhanced by sonication on
Bandlin-Sonerex (Bandelin, Berlin). UV spectra of karanjin for selecting the working wavelength of detection
were recorded on Cintra 5 UV-Visible spectrophotometer (GBC Scientific equipments, Australia).
METHADOLOGY:
A collected dried seed were crushed into powder and was taken into column weighed amount of powder as 200g.
Kept socking for about 12hours with the solvent Hexane so as to remove fat solubles, then after run with
Methanol and collect the methanol extract and kept for concentration through rotovapour so as to get the
methanol extract weight 22.36g. This methanol extract was treated with hexane to get Hexane solubles weighted
4.91g. remained Hexane insolubles was treated with Ethyl acetate to get Ethyl acetate solubles, EtOAc.50ml +
H2O 5ml was added and kept for steering on mechanical stirrer separate organic layer through separating funnel
collected Ethylacetate solubles, weighed 1.85g. which was analysed for the %purity by HPLC instrument. In the
same way kernels and shell were treated to get the ethylacetate solubles weighed 4.46g. & 0.59g. respectively to
find out %purity for the respective seed parts and reported the highest %yield of active constituent Karanjin.
FRACTIONISATION OF METHANOLIC EXTRACT:
Separation of hexane solubles from methanolic extract:
Hexane solubles were separated from methanolic extract by means of mechanical stirring. In this, the
methanolic extract was taken in hexane solvent and applied mechanical stirring. The traces of hexane solubles are
goes in to hexane solvent. This process was repeated by continuous replacement of saturated hexane with that off
fresh hexane solvent up to clear hexane solvent obtained. The hexane solubles were concentrated under vacuum,
by using Rota vapor, dried completely and weighed.
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Separation of ethyl acetate solubles from methanolic extract:
Ethyl acetate solubles were separated from methanolic extract by means of mechanical stirring. In
this, the methanolic extract was taken in ethyl acetate: water mixture (90:10) and applied mechanical stirring. The
ethyl acetate solubles goes in to ethyl acetate portion and remained polar solubles goes in to water portion in the
mixture. The ethyl acetate portion was separated from the water portion by using separating funnel. This process
was repeated by continuous replacement of saturated ethyl acetate with that off fresh ethyl acetate solvent up to
clear ethyl acetate solvent obtained. The ethyl acetate solubles were concentrated under vacuum, by using Rota
vapor, dried completely and weighed.
This was done for the different seed taken those are seed (dry), Kernal (dry), shell (dry), seed (fresh) &
kernel (fresh). The weights taken and the concentrated methanol, hexane and ethyl acetate solubles obtained dry
weights are given in below table no 1.
Table 1: Amount of Ethyl acetate solubles with different parts of P.glabra seed
S.No. Parts of P.glabra
seed taken 200g
Methanol
extract (g)
Hexane solubles
weight (g)
Ethylacetate solubles
weight (g)
1.
2.
3.
4.
5.
Seed (dry)
Kernel (dry)
Shell (dry)
Seed (fresh)
Kernel (fresh)
23.36
36.92
5.37
189
22.31
4.91
8.63
0.87
2.79
2.71
1.85
4.46
0.59
0.98
5.24
IDENTIFICATION OF FLAVONOIDS IN THE SEED EXTRACT OF PONGAMIA GLABRA:
Chemical tests:
a) Alcoholic solution of root extract reacts with freshly prepared ferric chloride Solution and given blackfish
green colour.
b) Alcoholic solution of root extract reacts with 10% lead acetate solution and given yellow precipitate.
c) Shinoda test: Alcoholic solution of root extract in the presence of magnesium ribbon and concentrated
hydrochloric acid given magenta colour.
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d) Alcoholic solution of root extract in the presence of galcial acetic acid and concentrated sulfuric acid
given green colour.
e) Alcoholic solution of root extract reacts with few drops of aluminum solution and given yellow colour.
PAST WORK RELATED TO PONGAMIA GLABRA
1. Quantification of Karanjin, tannin and trypsin inhibitors in raw and detoxified expeller and solvent
extracted Karanj (Pongamia glabra) cake[23]
2. Quantification of karanjin using high performance liquid chromatography in raw and detoxified karanjin
(Pongamia glabra vent) seed cake[24]
3. Determination of pongamol and karanjin in karanja oil by RP-HPLC[25]
EXPERIMENTAL PART:
Chromatographic Conditions:
The mobile phase consisting of methanol: water (85:15 %v/v). Prior to use, the mobile phase was de-gassed by
sonication. Between the samples the injection needle was washed with methanol. The mobile phase was pumped
through the system at a flow rate of 0.5ml/min. All experiments were carried out at ambient temperature of 23oC.
The UV wave length was set at 305nm. The retention time of karanjin was 4.561min. The optimized
chromatographic conditions of karanjin were shown in table 2.
Table 2: Optimized chromatographic conditions of karanjin
S.No. PARAMETERS
CONDITIONS
1.
2.
3.
4.
5.
Mobile phase
Stationary phase
Flow rate (ml/min.)
Run time (min.)
Column temperature (oC)
Methanol: Water
(85:15%v/v)
Waters LC-18
(150 x 4.6 i.d, 3µm.)
0.5
15
23
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6.
7.
8.
Volume of injection (µl)
Detection wavelength (nm)
Retention time of karanjin (min)
20
305
4.573
RESULTS:
METHOD VALIDATION:
1. SPECIFICITY:
To justify the purity of the compound FT-IR and 1H-NMR Spectrums were also attached in below for FT-
IR spectrum fig 5 and for 1H-NMR spectrum fig 6 were illustrated.
The HPLC chromatograms recorded for the crude extracts i.e., other peaks along with karanjin with in a
retention time range of 15min. and the standard pure karanjin fig.7 was compared the retention time for pure and
crude extracts from different parts of seed such as from seed (d), kernel (d), and for shell (d) in and for different
solvents used .The compounds are well separated from each other. Thus, the HPLC method presented in this
study is selective for karanjin quantification.
2. LINEARITY:
The regression analysis of standard concentrations of karanjin using weight regression analysis (weight +
1 / (concentration)2), the calibration curve was linear in the range as illustrated in fig 9. The mean ± standard
deviation (SD) for the slope, intercepts and correlation coefficient of standard curves (n=5) were calculated. The
regression data was shown in table 3
Table3: Linearity of calibration curves for karanj in.
1.
2.
3.
4.
5.
6.
60
48
37
24
12
06
14112
10864
9335
5991
2732
1405
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3. RECOVERY:
Percent recoveries of compounds from spiked blank were found and are represented as mean ± standard
deviation. For karanjin, at concentrations 6.3, 50, 100 & 200ng/ml. For pure karanjin, at a concentration of 25
ng/ml, the recovery was 95.10. The data of recovery studies of crude extracts shown in Table 4.
Table 4: Recovery studies of crude extract of karanjin.
Nominal
concentration
(ng/ml)
Measured concentration
(ng/ml) ± Standard
Deviation
Recovery
6.3
50
100
200
06.10 ± 0.05
50.10 ± 0.52
99.92 ± 3.01
200.1 ± 3.80
96.82
100.02
99.92
100.05
4. PRECISION & ACCURACY:
Intra – assay precision of the method was shown in Table 5. This was estimated by assaying the quality
control samples (6.3, 50, 100 & 200 ng/ml) five times in the same analytical run. The precision was less than 3.0
and the % relative error less than -3.0.
Inter – assay precision of the method was shown in table 6. This was estimated by assaying the quality
control samples (6.3, 50, 100 & 200 ng/ml) for replicate samples (n=5). The precision was less than 3.2 at all
levels meeting the acceptance criteria of ± 20% at LOQ and ± 15% at other levels.
Table 5: Precision (C.V.) and accuracy (relative error) of intra-day assay measurements of karanjin at UV detection 305 nm.
S.No. Nominal
concentration (ng/ml)
Measured concentration
(ng/ml) ± S.D.
% C.V.
1.
2.
3.
4.
6.3
50
100
200
06.10± 0.05
50.10 ± 0.52
99.92 ± 3.01
200.1 ± 3.80
0.893
0.998
3.009
0.184
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Table 6: Precision (C.V.) and accuracy (relative error) of inter – day assay measurements of karanjin at
UV detection 305 nm.
S.No. Nominal
concentration (ng/ml)
Measured concentration
(ng/ml) ± S.D.
% C.V.
1.
2.
3.
4.
6.3
50
100
200
6.01 ± 0.057
50.02 ± 0.59
98.95 ± 3.19
200.01 ± 3.98
0.945
1.102
3.210
1.981
5. LIMIT OF QUANTIFICATION & LIMIT OF DETECTION (LO Q& LOD)
It was found that below 5ng/ml, the back calculation values failed to meet the acceptance criteria. Hence
5ng/ml levels were five times injected. It was found that RSD was 2.75%. Accuracy, defined as the deviation
between the true values expressed, as a percentage was 3.9% at this concentration (5ng/ml). So 5ng/ml was
established as LOQ and 3ng/ml was established as LOD.
6. SYSTEM SUITABILITY
for system suitability, five replicates of standard samples were injected and studied the parameters like
theoretical plate number (N), tailing factor (T), resolution (R), relative retention time (α), high efficiency
theoretical plates (HETP), capacity factor (k´), plate per meter and peak symmetry of samples. These values are
mentioned in below table 7.
Table-7: System suitability parameters of karanjin.
S.No. Parameters Values
1.
2.
3.
4.
5.
Theoretical plates (N)
Tailing factor (T)
Resolution ( R)
Relative retention time (α)
HETP
2291
0.98
2.10
4.561
0.074
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6.
7.
8.
Capacity factor (K´)
Plates per meter
Peak symmetry
1.80
15163
1.2
7. ROBUSTNESS
As per the results of the percentage recoveries of karanjin was good under most conditions and did not
show any significant change when the critical parameters were modified. The components were well separated
under all the conditions carried out considering the modifications in the system suitability parameters and the
specificity of the method, as well as carrying the experiment at room temperature may conclude that the method
conditions are robust.
8. PURITY STUDIES OF KARANJIN FROM PLANT CRUDE EXTR ACTS
The purity study for different crude extracts was calculated according to the formulae given. The %purity
studies were given in the below table 8 for different parts of seed extracts and for different solvents given.
Table 8: Karanjin content in different parts of seed extract.
S.No. Part of seed from
P.glabra
MeOH extract
(grams)
EtOAc solubles of
MeOH extract (g)
% of
karanjin
1.
2.
3.
4.
5.
Seed (d)
Kernel (d)
Shell (d)
Seed (f)
Kernel (f)
11.20%
18.46%
2.68%
11.54%
10.93%
0.92%
2.18%
0.32%
1.99%
2.55%
38.35
53.52
12.35
38.25
14.26
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Here the graphs of the developed methods are given below:
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DISSICUSION:
The Chromatographic method was optimized by changing various parameters, such as pH of the mobile
phase, the separation of peaks are dependent on the percentage of methanol. Methanol was used instead of
acetonitril to shorten tailing of karanjin. In case of acetonitril, the karanjin show more tailing, even though the
retention time is less than the methanol.
Under the presently prescribed conditions, the recovery of karanjin was found to be 95.102%, a very low
concentration.
Till now there is no single method was developed for the determination of karanjin with out buffer. So
this method is useful for determining the %purity of karanjin in crude extracts.
The observation of C.V. less than 4.0 for both inter-day and intra-day measurements also indicates the
high degree of precision.
In the present method, we have established a linearity range of 6 – 60 µg/ml; this linearity range covers all
the strengths of karanjin. Hence this method can be applied for quantifying the low levels of karanjin in extracts
and other pharmacokinetics studies if necessary.
CONCLUTION
The aim of the present study was to develop simple, fast and sensitive HPLC methods for the estimation
of Karanjin from P. glabra few analytical methods are reported in literature, but those methods have some
drawbacks like gradient elution, long run time, less resolution and lack of sensitivity like precision and accuracy.
Further more for karanjin there was no proper documentation and validation, which are crucial for analytical
method here, it was mentioned clearly.
The new reverse-phase high performance liquid chromatographic methods were developed and validated
for the determination of Karanjin in crude extreact & method was developed.
The method described herein is simple validated assay procedures that can readily be used in any
laboratory for the quantitative determination of crude extracts. Analytical figures of merit demonstrated during
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the method validation protocol compare well with those of known methods for the determination of crude
extracts from different parts of seed. The assay procedure was simple with satisfactory precision and accuracy in
terms of relative error. We believe that this method fulfills requirements for determining the crude karanjin and
for %purity studies from different extracts of the plant parts.
Practically, low cost of single analysis is central features of routine laboratory and the herein described
RP-HPLC method, because it uses low cost of solvents and is easily affordable by clinical laboratories equipped
with standard high performance liquid chromatographic systems.
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Corresponding Author:
Vejendla.Ravikumar*,
Departmant of Pharmacy,
Sri Indu Institute of Pharmacy,
Hyderabad.
Email: [email protected]